Objective. Sasang typology is a traditional Korean medicine based on the biopsychosocial perspectives of Neo-Confucianism and utilizes medical herbs and acupuncture for type-specific treatment. This study was designed to develop and validate the Sasang Personality Questionnaire (SPQ) for future use in the assessment of personality based on Sasang typology. Design and Methods. We selected questionnaire items using internal consistency analysis and examined construct validity with explorative factor analysis using 245 healthy participants. Test-retest reliability as well as convergent validity were examined. Results. The 14-item SPQ showed acceptable internal consistency  and test-retest reliability (r = .837). Three extracted subscales, SPQ-behavior, SPQ-emotionality, and SPQ-cognition, were found, explaining 55.77% of the total variance. The SPQ significantly correlated with Temperament and Character Inventory novelty seeking (r = .462), harm avoidance (r = −.390), and NEO Personality Inventory extraversion (r = .629). The SPQ score of the So-Eum (24.43 ± 4.93), Tae-Eum (27.33 ± 5.88), and So-Yang (30.90 ± 5.23) types were significantly different from each other (P < .01). Conclusion. Current results demonstrated the reliability and validity of the SPQ and its subscales that can be utilized as an objective instrument for conducting personalized medicine research incorporating the biopsychosocial perspective. Personality represents a person's unique pattern of behavior, emotionality, and way of cognition, in addition to specific body features that interact to determine individual's adaptation to the environment , 2. DiseThe traditional Korean Medicine adopted such perspectives and proposes a systematic medical typology  within tExamination of corresponding Western personality traits in Sasang typology began with the Minnesota Multiphasic Personality Inventory in 1992 and has evolved to the use of various objective tools such as the 16-Personality Factor (16-PF), Myers Briggs Type Indicator (MBTI), State Trait Anxiety Inventory (STAI), Beck Depression Inventory (BDI), Eysenck Personality Questionnaire (EPQ), NEO-Personality Inventory (NEO-PI), and Temperament and Character Inventory (TCI). The theoretical and descriptive similarity of Sasang typology with the Western tradition of personality including Hippocrates and Eysenck has been suggested from these results . More spThere have been several reports on the influence of personality traits on health , 18. ForStudy participants were 245 students from the School of Korean Medicine at Pusan National University and the College of Oriental Medicine at Wonkwang University. This study was approved by the Institutional Review Board (IRB) of the Pusan National University School of Korean Medicine (KCRC IRB 2010-01). All participants gave written consent for the assessments. The mean age of the 245 participants  was 29.01 ± 5.67 (range 21–46).Since 20 participants did not receive their Sasang type classification based on the Questionnaire for Sasang Constitution Classification II (QSCCII), SPQ was examined with the remaining 225 participants . The mean age of each Sasang type based on the QSCCII was 29.58 ± 6.31 for the So-Yang type , 28.26 ± 5.47 for the Tae-Eum type , and 28.79 ± 5.52 for the So-Eum type .n = 225) test-retest reliability of the SPQ and its subscales were analyzed (n = 45).SPQ item selection using internal consistency, explorative factor analysis using parallel analysis, convergent validity using correlation analysis between SPQ and SPQ subscales, TCI, and NEO-PI were performed   is a 140The NEO-PI-R is a 60-item self-report inventory on a 5-point scale  based on factor analysis designed to assess a wide spectrum of individual differences, including the universal, stable, and consistent big five structures of neuroticism, extraversion, openness to experience, agreeableness, and conscientiousness . CronbacThe questionnaire items for the internal consistency analysis were selected from the 42 items collected from the information database of the Sasang typology project (2006-2007) and an online questionnaire development project (2005–2007) of Korea Institute of Oriental Medicine. These questionnaire items were based on Sasang typology theory and descriptions of clinical characteristics of each Sasang type . Items tEach item consists of two opposite words describing a particular personality trait, and participants must respond on a 3-point scale. This response system follows the typical way in which Sasang-type classification is made in clinical practice. For example, the participant can respond as “delicate (= 1),” “average/middle (= 2),” or “tough (= 3)” to the question “Do you have a delicate or tough personality?” The So-Eum type was expected to have a lower score while the So-Yang type evidence higher score on this particular SPQ item based on the consideration of previously reported clinical descriptions of each Sasang type .A review board composed of three traditional medical doctors with more than 5 years of clinical experience and one licensed medical specialist in Korean Sasang typology then selected 15 items that were judged to have a high association with Sasang typology and which may represent typical features of each Sasang type.The internal consistency of the preliminary 15-item SPQ was examined, after which one item that revealed a low correlation and judged to have low clinical importance was deleted from the final version of the SPQ. That is, the 5th questionnaire item (Q5) was deleted following preliminary item analysis, since the Cronbach's alpha was able to be increased up to 0.817 when deleted. Although the 11th questionnaire item (Q11) was found to increase questionnaire's internal consistency to 0.816 if deleted, this item was decided to be included following panel discussion due to having clinically significant contribution to explaining emotionality of Sasang typology. Therefore, the final 14-item SPQ  showed aThe 14 items were subjected to explorative factor analysis to examine the possible structure of the SPQ using principal axis extraction and Promax rotation with an eigenvalue over 1.0 as the criteria. We also performed parallel analysis to get the right factor numbers using Monte Carlo PCA for Parallel Analysis which estimates an average distribution of eigenvalues based on a random process that can be compared to the calculated distribution , 34. TheThe test-retest data for the reliability examination over a period of one month was analyzed with correlational analysis. The reliability of SPQ and its subscales were determined with Pearson's correlational coefficients. The criterion validity of the SPQ and its subscales were examined by comparing the relationship with the well-established TCI and NEO-PI. We used Spearman's correlational coefficients exceeding the minimum acceptable value of 0.3.The SPQ and its subscales  were subjected to analysis of variance (ANOVA) and profile analysis such as test of parallelism and flatness to examine its effectiveness in representing temperament differences between the Sasang-type groups based on “QSCC II classification”. Demographic differences between Sasang-type groups were tested using ANOVA for continuous variable (age) and Fisher's exact tests for categorical variables (gender).In addition, we compared the differences of each Sasang-type group on the TCI and NEO-PI to examine if the participants of this study have similar characteristics as in previous studies. ANOVA was conducted to test between group differences in TCI and NEO-PI scores, and Scheffé was used for post hoc analysis, and the profile analysis was performed to test the difference of the TCI and NEO-PI profiles for each Sasang-type group .P value of 0.01 and 0.001 were used for significance.The data are presented as means and standard deviations or frequency with percentage. All analyses were conducted using PASW Statistics 18.0 for Windows  and Promax rotation procedure with SPQ 14 items  yielded The first factor, which accounted for 31.5% of the variance, included behavioral components of personality such as “do you consider yourself passive or proactive?” Factor 2, which accounted for 14.9% of the variance, included emotionality component of personality such as “are you relatively patient or impatient?” Factor 3, which accounted for 9.4% of the variance, included personality component related to cognition/decision-making, such as “do you have a delicate or a tough personality?”r = 0.827), SPQ-E (r = 0.649), and SPQ-C (r = 0.805). SPQ-B showed significant correlation with SPQ-E (r = 0.301) and SPQ-C (r = 0.541).The three factors were defined as follows. SPQ-behavior (SPQ-B) measures the behavioral component of personality (passive versus active); SPQ-emotionality (SPQ-E) measures the emotionality component of personality (static versus dynamic); finally, SPQ-cognition (SPQ-C) measures the cognition/decision-making or cognitive component of personality (meticulous versus easy-going). The items comprising each subscale are outlined in The test-retest reliability of the SPQ and its subscales were analyzed with Pearson's correlational analysis. Overall test-retest reliability was found to be 0.837, and reliabilities of the three subscales of SPQ-B, SPQ-E, and SPQ-C were 0.830, 0.748, and 0.798, respectively .The criterion validity of the SPQ was examined with the TCI and NEO-PI. SPQ and its subscales showed significant relations with the TCI and NEO-PI .df = 2;  P = 0.061) and age .The biopsychological characteristics of the participants in this study were analyzed to determine whether the SPQ demonstrates temperament differences between Sasang-type groups and to confirm whether the TCI and NEO-PI scores replicate findings of previous studies . First odf = 2, F = 47.789, and P < 0.001). As for the parallelism of SPQ subfactor profile with the interaction of Sasang type were significantly different .The SPQ subscales  of each Sasang type showed significantly different profiles . The proF = 28.157;df = 2,213;P < 0.001), SPQ-B , SPQ-E , and SPQ-C  between Sasang types were demonstrated with ANOVA. Post hoc analysis showed that the SPQ of the So-Yang type (30.90 ± 5.23) was significantly higher than that of the Tae-Eum type (27.33 ± 5.88) (P = 0.002), and that of the Tae-Eum type was significantly higher than that of the So-Eum type (24.43 ± 4.93) (P = 0.005), while SPQ score of the So-Yang type was significantly higher than that of the So-Eum type (P < 0.001). Post hoc analysis showed that the SPQ-B of the So-Yang type (11.62 ± 2.27) was significantly higher than that of the Tae-Eum type (9.91 ± 2.93) and the So-Eum type (8.90 ± 2.44) , respectively. Post hoc analysis showed that the SPQ-E of So-Yang type (8.67 ± 2.51) is significantly higher than those of Tae-Eum type (7.23 ± 2.32) and So-Eum type (7.23 ± 1.97) , respectively. Post hoc analysis showed that the SPQ-C of the So-Eum type (8.30 ± 2.45) is significantly lower than those of Tae-Eum type (10.19 ± 2.62) and So-Yang type (10.62 ± 2.33) , respectively.Significant differences on the SPQ . As for the parallelism of TCI temperament profile with the interaction of Sasang types, they were significantly different . The TCI temperament  of each Sasang type exhibited significantly different profiles . The proF = 7.315, df = 2,222, and P < 0.001), TCI harm avoidance , and TCI reward dependence  between Sasang types were demonstrated with ANOVA. Post hoc analysis showed that the TCI harm avoidance of the So-Yang type (34.92 ± 10.06) and Tae-Eum type (35.88 ± 12.76) was significantly lower than that of the So-Eum type (41.57 ± 11.86) .The significant differences on the TCI novelty seeking  between Sasang types were demonstrated with ANOVA. Post hoc analysis showed that the NEO-PI conscientiousness of the So-Yang type (48.39 ± 7.49) was significantly lower than that of the So-Eum type (54.58 ± 7.63) (P = 0.006). The NEO-PI extraversion of the So-Yang type (55.00 ± 10.04) was higher than that of So-Eum type (49.56 ± 9.60), but not statistically significant.The NEO-PI subscales did not show significant profile differences between Sasang types . HoweverPersonalized medicine revolves around the use of safe and effective medical treatment best fit to specific patients, and this theme has been a major concern in both Western orthodox and traditional Eastern medicine. Sasang typology founded in Neo-Confucianism is a temperament-based personalized medicine using acupuncture and medical herbs . In ordeReliability and validity examination of the 14-item SPQ demonstrated that the SPQ shows the benefit of multifaceted structural analysis of Sasang typology across the domains of behavior, emotionality, and cognition with acceptable reliability and stability . FurtherIt was found that the SPQ measures three facets of personality associated with Sasang typology, namely, behavior, emotionality, and cognition. More specifically, the So-Eum type scored relatively lower on the SPQ such that its personality can be characterized as passive (behavior), static , and meticulous (cognition). In contrast, the personality profile of the So-Yang type can be characterized as active (behavior), dynamic , and easy-going (cognition) as indicated by a higher score on the SPQ. From the perspective of traditional Korean medicine, such characteristics of the SPQ subscales reflect the basic theoretical foundation of Eastern philosophy that the smaller change in behavior, emotionality, and cognitive characteristics of the SPQ mirror the Yin (So-Eum type), while greater change mirrors the Yang (So-Yang type). : Tae-Eum : So-Yang axis as suggested in previous studies [If we sum up these results, SPQ is a reliable and objective psychological measurement of the So-Eum studies  and has  studies , which iThe psychological structure of the SPQ subscales was compared with existing validated psychological instruments using correlational analysis. The results indicated that the SPQ and its subscales are generally in the same direction as the TCI. However, it was found that the SPQ-emotionality subscale demonstrated a positive correlation with NEO-PI neuroticism, but the SPQ-behavior and SPQ-cognition subscales showed negative correlations . The NEOThis disparity could be the reason why current and previous studies  failed tAs for the behavioral, emotional, and cognitive facets of the Sasang personality in this study, the manner in which an individual will evaluate a particular situation or environment will be determined by the individual's emotional state, which will in turn be expressed in various corresponding behavioral forms judged to be suitable to the situation or event. For example, the positive affect-negative affect dimension has emerged as an important dimension of an individual's emotional experience, such that self-estimate of such affect results in expressions of mood that correspond to measures of personality and emotionality, in addition to making possible predictions of cognitive performance . The assSince there are insufficient studies examining the biopsychological basis and its medical value in different medical typologies, more scrupulous and practical studies are needed. Current studies with conventional Western medicine have reported that specific temperaments make a person predisposed to certain disease and affect its prognosis. For example, although much disagreement still exists, the link between cancer and type C personality was suggested wherein a personality that suppresses emotions and shows difficulty in coping with stress , and a pThe SPQ and its subscales  showed three facets of Sasang personality and provide further rationale for how one's personality may affect health in clinical health psychology studies , 49. TheThe relationship between stress and anxiety (neuroticism) has gained much interest in recent days. Predisposition of the HPA axis related to the stress response, the functioning of the stress-related response by the interleukin and natural killer cell have also been proposed as potential mediators in the stress-response cycle . The hosThis study has several limitations that may affect generalizability. First, this study should be repeated with a larger and more balanced sample size. Although the TCI and NEO-PI scores of each Sasang-type groups in this study were not significantly different compared to previous studies , 38, theSecond, SPQ-behavior, SPQ-emotionality, and SPQ-cognition were extracted as the subscales of SPQ, and these should be validated with diverse psychological instruments alongside with their stability. In particular, as the SPQ-emotionality correlated with NEO-PI neuroticism while, SPQ, SPQ-behavior, and SPQ-cognition negatively correlated with NEO-PI neuroticism, other assessments much are examined to see whether such patterns continue to hold. The reason for distinct differences in emotionality or neuroticism dimension between Western psychology and Sasang typology should be analyzed to determine if such differences result from cultural or philosophical disparities, clinical experience, personality construct, or other factors .Third, the possibility of response bias, or the tendency of participants to provide socially acceptable responses, cannot be ruled out, especially as they must self-assess using the SPQ. However, this may have been minimized due to the forced-choice nature of the SPQ and the fact that participants were unaware of its scoring method. In addition, the relatively high test-retest reliability score of the SPQ and the replication of theory-driven association with the NEO-PI and TCI suggest that the likelihood of response bias may have been reduced in the present study. Last but not least, the clinical usefulness of SPQ should be substantiated using clinical samples. Although the psychological and biometric profiles of each Sasang type in this study using the QSCCII are a replication of previous clinical studies , 38, theIn conclusion, the Sasang Personality Questionnaire which can objectively measure the psychological personality basis of Sasang typology was developed and validated with Western psychometric instruments in this study. With further clinical investigation, the SPQ may serve as a solid foundation for personalized medicine with medical herbs and acupuncture by providing a biopsychosocial typology perspective.


It has been questioned if the old native Norwegian sheep breed, Old Norse Sheep , normally distributed on coastal areas where ticks are abundant, is more protected against tick-borne infections than other Norwegian breeds due to a continuously high selection pressure on pasture. The aim of the present study was to test this hypothesis in an experimental infection study.Anaplasma phagocytophilum (similar to GenBank accession number M73220). The experiment was repeated for two subsequent years, 2008 and 2009, with the use of 16 lambs of each breed annually. Ten lambs of each breed were inoculated intravenously each year with 0.4 ml A. phagocytophilum-infected blood containing approximately 0.5 × 106 infected neutrophils/ml. Six lambs of each breed were used as uninfected controls. Half of the primary inoculated lambs in each breed were re-challenged with the same infectious dose at nine (2008) and twelve (2009) weeks after the first challenge. The clinical, haematological and serological responses to A. phagocytophilum infection were compared in the two sheep breeds.Five-months-old lambs of two Norwegian sheep breeds, Norwegian White (NW) sheep and Old Norse (ON) sheep, were experimentally infected with a 16S rRNA genetic variant of A. phagocytophilum.The present study indicates a difference in fever response and infection rate between breeds of Norwegian sheep after experimental infection with Although clinical response seems to be less in ON-lambs compared to NW-lambs, further studies including more animals are needed to evaluate if the ON-breed is more protected against tick-borne infections than other Norwegian breeds. Anaplasma phagocytophilum (formerly Ehrlichia phagocytophila) is an endemic disease of sheep in tick (Ixodes ricinus) infested areas of Norway. Natural infection with A. phagocytophilum has been reported in humans and a variety of domestic and wild animal species [Staphylococcus spp. [Pasteurella (Mannheimia) septicaemia [Tick-borne fever (TBF) caused by the bacteria  species . TBF is cus spp. , and Pasticaemia ,4. Complticaemia , reducedticaemia , impaireticaemia , and redticaemia . The infticaemia .A. phagocytophilum infection is widespread along the coast of southern Norway. However, clinical TBF was only diagnosed in half of these seropositive sheep flocks [A. phagocytophilum may co-exist in the same sheep flock and even in the same animal [A serological survey in sheep indicated that p flocks . The reap flocks ,10. Basee animal . The geoA. phagocytophilum infection [A. phagocytophilum infection in two Norwegian sheep breeds.The management of sheep flocks may vary considerably in Norway. While the dominant Norwegian sheep breed, Norwegian White (NW) sheep, are normally housed indoors during the winter season and treated regularly against ticks and gastro-intestinal parasites, the Old Norse (ON) sheep breed can be on pasture the whole year around with limited parasitic treatment . A Britinfection . Based oA. phagocytophilum. Based on partial sequencing of the 16S rRNA gene, a variant of A. phagocytophilum was found in one lamb, similar to GenBank accession number M73220[Blood samples were collected from a flock of Norwegian sheep known to be infected with er M73220. Both EDI. ricinus and were kept indoors during the whole experimental period of four to five months. The lambs of each breed were grouped in infected and controls, according to equal distribution of sex and mean live weight. Ten lambs of each breed were inoculated intravenously (day 0) with 0.4 ml of the above mentioned DMSO-stabilate of A. phagocytophilum using one aliquot for each breed. The infectious blood contained approximately 0.5 × 106 infected neutrophilic granulocytes/ml. Six lambs in each group were left as uninfected controls. Nine weeks (2008) and twelve weeks (2009) after the primary inoculation, five of the earlier inoculated lambs of each breed were selected by random sampling and re-challenged with the same infectious dose of the homologous variant.A total number of 64 lambs, 5 months old, were used in this trial, 32 lambs of the NW-breed and 32 of the ON-breed. The experiment was approved by the National Animal Research Authority (Norway). The study was conducted for two following years, 2008 and 2009, with the yearly use of 16 lambs of each breed. The ON-lambs came from two different sheep herds in Rogaland county, lambs from one herd were used each year, while all NW-lambs belonged to the experimental sheep flock at the Department of Production Animal Clinical Sciences. All ON-lambs were adapted and housed at the department three to four months before start of the experimental period. None of the lambs had previously been exposed to pasture with The lambs were observed at least twice a day. Rectal temperature was measured daily in all lambs throughout the experiment. The incubation period was defined as the period between inoculation and the first day of fever (≥ 40.0°C), and the duration of fever was recorded as the number of days with elevated body temperature (≥ 40.0°C). The magnitude of fever of each lamb was estimated from the area of plots of daily temperature on 5 mm grids and calculated according to the Trapezium Rule . For thi®, Bayer) and blood smears were prepared and stained with May-Grünwald Giemsa. Four hundred neutrophils were examined on each smear by light microscopy, and the number of cells containing Anaplasma inclusions was recorded. The infection rate (percentage of infected neutrophils) was calculated. The body weight of all lambs was measured weekly, during the whole experimental period. The average live weights (± SD) of the lambs at the start of the experiment were 52.8 ± 6.86 (NW-2008), 44.9 ± 6.10 (NW-2009), 28.8 ± 4.10 (ON-2008), and 21.3 ± 3.44 (ON-2009), respectively.Daily EDTA blood samples were collected during the fever period, and on a weekly basis after the fever had subsided. In addition, EDTA blood samples were later collected from individual lambs when intermittent rectal temperatures above 40.0°C were recorded. Total and differential leucocyte counts were determined electronically  were analysed for Anaplasma -DNA by PCR [260 spectrophotometry . The samples were diluted 1:100 before PCR analysis.In order to investigate A by PCR . Brieflymsp2(p44) expression site. The primers were manufactured by TIB Molbiol (Germany). A Light Cycler 480 instrument (Roche) was used for the real-time PCR analysis. A total of 96 well white plates were loaded with a reaction mix consisting of 0.5 μl (5 μM) ApMSP2 252 primer, 0.5 μl (5 μM) ApMSP2 459 primer, 1.5 μl RNAse free H2O, 5 μl LightCycler 480 DNA SYBR Green I Master and 2.5 μl sample. Plates were sealed by sealing foil and centrifuged at 1200 rpm for two minutes. Samples and non-template controls were run in duplicates on each plate. The Cq values (quantification cycle) were determined by the 2nd derivative maximum method and verified by melting point analysis (Tm) [The primers were Ap MSP2 252 5' ACAGTCCAGCGTTTAGCAAGA and Ap MSP2 459 5' CACCACCAATACCATAACCA, amplifying a product of 208 bp at the N-terminal hyper variable region of the sis (Tm) .Ehrlichia equi [A. phagocytophilum (formerly E. equi) antigen . Bound antibodies were visualized by fluorescein-isothiocyanate (FITC)-conjugated rabbit-anti-sheep immunoglobulin . Sera were screened for antibodies at dilution 1:40. If positive, the serum was further diluted and retested. A titre of 1.6  or more was regarded positive.Sera were collected at days 0, 7, 14, 21, 28, 42, and 63  after each inoculation and analysed using an indirect immunofluorescence antibody assay (IFA) to determine the antibody titre to hia equi ,16. Briet test was used to analyse clinical, haematological and serological variables. A P value of < 0.05 was considered significant.Statistical calculations were done using Statistix, version 4.0 , and a two-sample A. phagocytophilum, while infected ON-lambs showed no signs of clinical illness.In general, a one to two days period with reduced appetite was observed in the NW-lambs infected with In 2008, all inoculated lambs from both groups developed fever. Significant differences in duration of fever and magnitude of fever were recorded between the two sheep breeds Table . In addiAfter re-challenge on day 63 (2008), clinical signs were not observed, and only one lamb of each breed reacted with a detectable infection rate , duration of neutropenia (7 days) and a serological response . All the other four ON-lambs also reacted with fever of 1-2 days duration, cytoplasmic inclusions (max. infection rate: 6-56%), and neutropenia (duration: 3-5 days) after re-challenge. A significant increase in antibody titre (p < 0.05) was observed in the re-challenged lambs compared with the unchallenged lambs on days 98, 105 and 112, respectively (data not shown). In contrast, the five NW-lambs that were re-challenged on day 84, did not react with a clinical response, cytoplasmic inclusions or a titre increase . The infection rate was significantly different only on days 3 and 4 (p < 0.001). However, significant differences were observed in the antibody titres on days 14, 21, 28, 42 and 63 (p < 0.001). Significant differences in the antibody responses were also observed between the ON-lambs in 2008 and 2009, i.e. on days 14, 21, 28, 42 and 63 (p < 0.01), respectively. In addition, variation in clinical and haematological responses and infection rates were observed within the ON-breed, such as in the maximum temperature (p < 0.05), magnitude of fever (p < 0.01), duration of neutropenia (p < 0.01) and weight gain (p < 0.001) (two-sample t test).Comparison of results from 2008 and 2009, showed no difference in clinical signs or haematological reaction between the NW-lambs  for the first nine weeks in the infected and control lambs were 180 ± 21.2 g/d (NW- infected), 204 ± 35.9 g/d (NW-control), 155 ± 47.8 g/d (ON-infected), and 164 ± 44.5 g/d (ON-control), respectively. No significant differences between the infected and control lambs within each breed were observed.Similarly, the mean daily weight gains (± SD) for the first twelve weeks in the infected and control lambs in 2009 were 137 ± 30.0 g/d (NW-infected), 165 ± 16.1 (NW-control), 78.1 ± 29.0 g/d (ON-infected), and 105 ± 18.9 (ON-control), respectively. No significant differences between the infected and control lambs within each breed were observed.A. phagocytophilum infection [In the present study, only a limited number of animals of each breed were included. Earlier experimental studies in NW-lambs indicate a variation in clinical, serological and haematological reactions to an nfection . In ordeA. phagocytophilum infection were detected in the two sheep breeds. In 2008, no differences in the haematological and the serological reaction were observed. In contrast, marked differences were observed in 2009, where seven ON-lambs reacted with a prolonged incubation period and no evidence of clinical response in three lambs. Breed differences in response to A. phagocytophilum have, as mentioned earlier, been shown among British sheep breeds. Blackface sheep reacted less severely to tick-borne fever than other breeds and their crosses, however this seems not entirely attributable to past ancestral exposure to the pathogen [Significant differences in the clinical reaction and infection rate to a primary pathogen . The reaA. phagocytophilum group have been reported, but the titre response to a heterologous variant is normally less than against a homologous variant [E. equi was the only antigen available for use in the present study.A significant difference in the antibody titre response was recorded between the two years. This may be due to different batches of antigen used, since the sensitivity of the IFA-test may vary between batches . The sensitivity of the present antibody test may have been increased by use of a more proper antigen, i.e. an ovine variant of the bacterium. Strong serological cross-reactions between all members of the  variant . UnfortuIn contrast to NW-lambs, marked differences in clinical and haematological reactions were observed between years in the ON-lambs. The reason for these differences among ON-lambs is unknown. The animals were from two different geographical areas with no direct relationship, and the lambs had been adapted to the same experimental conditions for several months. In addition, earlier studies indicate that the individual diversity is not significantly different within the two sheep breeds involved .A. phagocytophilum. However, an innate immune response in infected lambs may be inefficient since A. phagocytophilum have lost all genes for synthesis of LPS and most genes for biosynthesis of peptidoglycans [In 2009, seven ON-lambs reacted with a long incubation period, a low maximum temperature and a short duration of fever . These roglycans ,20.A. phagocytophilum inoculum, however, the incubation period may be longer with a low infection dose. As little as one A. phagocytophilum infected cell is enough to transmit the infection [6 infected neutrophils, which should be more than sufficient to create a clinical reaction. However, reduction of the infectious dose in the aliquot used during freezing and thawing cannot be excluded.Earlier studies indicate that the amplitude of clinical and haematological reaction is independent of the dose of the nfection . In the Anaplasma-DNA was not detected in the three non-responsive lambs during the first fourteen days. The detection threshold of real-time PCR used was 10 copies of DNA [A. phagocytophilum.Most of the inoculated blood used in 2009 must have been infectious. However, if a small infection dose in the thawed aliquot was divided into ten infectious doses, some of these batches may have lacked infectivity. This statement is supported by the fact that s of DNA . These tA. phagocytophilum infection will have a negative effect on the autumn live weight of lambs [In the present study, the daily weight gain in lambs varied between the two years. This variation may be due to the quality of the roughage involved. Unfortunately, the quality of the silage used was not measured. The daily weight gain between the infected and non-infected animals of each breed was not significantly different. One study indicates that an early of lambs . HoweverA. phagocytophilum infection varies and that sheep may resist homologous challenge for a period from a few months to more than one year [A. phagocytophilum involved [Resistance to experimental re-infections rises with increasing frequency of challenge . Earlierone year ,24-26. Iinvolved .A. phagocytophilum infected ON-lambs are more resistant to other infectious agents compared to NW-lambs needs further investigation, since factors such as management and other external stress factors may be important to the outcome of infection [Earlier investigations have shown that lambs are susceptible to secondary infections, especially in the bacteraemic and neutropenic periods of TBF ,27-29. Infection .A. phagocytophilum. Whether these differences are due to genetic resistance that can be implemented in a breeding programme to increase the resistance against TBF, needs further exploration. However, studies including more animals are needed to evaluate if the ON-breed is more protected against tick-borne infections than the NW-breed. In addition, analyses of diseased and fatal cases of TBF must be performed in order to elucidate differences in pathogenicity and protective immunity between Norwegian sheep breeds.The present results indicate a difference in fever response and infection rate between breeds of Norwegian sheep after experimental infection with The authors declare that they have no competing interests.SS, LS, IO and HS have designed the experimental study. SS performed the experimental study, carried out the statistical analysis and drafted the manuscript. EGG carried out the molecular genetic analysis. KS performed the serology. All authors read and approved the final manuscript.


Lymnaea. A training procedure comprising 5 repeated pairings of a conditional stimulus , with an unconditional stimulus , over a 4-day period resulted in an enhanced memory formation than 10 CS-US repeated pairings over a 2-day period or 20 CS-US repeated pairings on a single day. Backward conditioning (US-CS) pairings did not result in conditioning. Thus, this taste avoidance conditioning was CS-US pairing specific. Food avoidance behavior was not observed following training, however, if snails were immediately subjected to a cold-block (4°C for 10 min). It was critical that the cold-block be applied within 10 min to block long-term memory (LTM) formation. Further, exposure to the cold-block 180 min after training also blocked both STM and LTM formation. The effects of the cold-block on subsequent learning and memory formation were also examined. We found no long lasting effects of the cold-block on subsequent memory formation. If protein kinase C was activated before the conditioning paradigm, snails could still acquire STM despite exposure to the cold-block.The present study investigated the optimal training procedure leading to long-lasting taste avoidance behavior in  Our understanding of the causal neuronal mechanisms that underlie learning and the subsequent formation of long-lasting memory has been greatly advanced by studies of simpler invertebrate model systems. Here we investigated the critical time periods following classical conditioning training during which memory formation can be interrupted using a reversible cold-block technique (cooling-induced amnesia). Knowledge of the critical time periods for consolidation is crucial to elucidate the molecular events that underlie memory.To establish taste avoidance conditioning (TAC), we paired sucrose application , which elicits a feeding response, with a tactile stimulus to the head , which evokes a withdrawal response. The withdrawal response is incompatible with feeding, and thus the feeding response elicited by the CS is immediately terminated. After a number of CS-US pairings, the CS no longer elicits feeding. If the order of the pairing is reversed , the CS continues to elicit feeding behavior .Cooling to less than 5 °C for a short period of time (tens of minutes) reversibly blocks the formation of long-term memory (LTM) following conditioning if performed within a short period of time following training –7. This The canonical view of memory formation is that learning is a serial process beginning with short-term memory (STM), which persists for minutes, followed by intermediate-term memory (ITM), which persists for a few hours, and then LTM, which persists for days to week to years . STM doeDrosophila, Aplysia, Lymnaea, Apis) preparations have demonstrated that ‘spaced’ training results in a longer lasting LTM than does ‘massed’ learning [Lymnaea that spaced or distributed training was more effective than massed training in both classical conditioning [Studies performed in both mammalian (human and rodent) and invertebrate . LTM requires both new protein and mRNA synthesis, which occurs following prolonged activation of PKA induced by the training procedure that produces LTM. The prolonged activation of PKA causes it to translocate from the cytoplasm of the neuron to the nucleus. In the nucleus, PKA phosphorylates cyclic AMP response element binding protein 1 (CREB-1). CREB-1 is a transcription factor, and its phosphorylation stimulates mRNA synthesis. Downstream from CREB-1 are the immediate response genes, including the CCAAT box enhancer-binding protein. Activation of the immediate response genes, in turn, stimulates the transcription of downstream genes that trigger long-term structural changes that are thought to be the morphologic correlates of LTM [Lymnaea [In s of LTM –31. A si[Lymnaea .Lymnaea TAC [Hermissenda crassicornis, rodents, and rabbits [Hermissenda, the PKC modulator, bryostatin (Bryo) enhanced STM by pathways involving PKC-initiated membrane protein phosphorylation [Hermissenda [Lymnaea TAC memory.A more recent study using a similar training procedure implicated the involvement of a molluscan insulin receptor in memory formation in naea TAC . PKC maynaea TAC ,35. For  rabbits –39. In Hrylation ,41. In amissenda ,42–44. HHere we first examined whether spaced training is more effective than massed training for TAC. Although we hypothesized that space training would be more effective, taste aversion may be differentially affected because in some taste aversion training procedures, the CS and US can be separated by hours and still be effective  . Second,Lymnaea stagnalis, with shell lengths of 20-25 mm, were maintained at 22°C in well-aerated fresh water on a 12-h light: 12-h dark cycle (on at 08:00). Snails larger than 20 mm in shell length are capable of classical conditioning [Laboratory-reared fresh water pond snails, itioning . AnimalsThe Plexiglas experimental container (diameter: 60 mm and height: 20 mm) had a perfusion system with an inlet and outlet from which the solution inside the container could be entirely replaced within 30 s (rate: 250 ml/min). The container held 10 ml pond water in which snails were kept. The conditioning response, i.e., feeding response, was readily observed by a mirror placed under the container.p<0.05) lower than that of the initial pre-test, i.e. that of the first day pre-test. Backward conditioning with a tactile stimulus presented to the head (US) followed by sucrose application (CS) was performed to confirm whether TAC is CS-US temporal specific.The conditioning procedure used was identical to that reported by Kawai et al (2004). In brief, snails were allowed to acclimatize for 10 min at 22°C in well-aerated pond water in the training container. Following acclimatization, the feeding response  to the CS (sucrose) was recorded. The CS (1 ml of 100 mM sucrose) was applied directly to the lip of the animal with a 1-ml syringe. Immediately following the sucrose application, the number of bites per minute (‘feeding response’) was tabulated for 1 min. A tactile stimulus (US) was then applied to the animal’s head using a hand-held Plexiglas rod. The stimulation was strong enough to always evoke a whole-body withdrawal response, as well as termination of repetitive mouth opening and closing (feeding). This was the pre-conditioning test (pre-test). After 10 to 15 min, the snails received varying numbers of CS-US pairings separated by 5-s intervals applied using a 1-min intertrial interval. The snail typically required less than 1 min to recover from the US. An immediate post-conditioning test, was applied at least 10 min or later after conditioning, was performed following the paired CS-US presentations. In this test only the CS was delivered. The immediate post-conditioning test was used to determine whether STM had formed. We defined STM as being present if the feeding response after conditioning (i.e. the post-test) was significantly suppressed in comparison with pre-test response on that day. We also tested the response to the CS 24 h and 48 h after training. The 24-h and 48-h post-conditioning tests enabled us to determine whether LTM had formed. Our definition of LTM was that the feeding score in the 24 or 48 h post-test was significantly (A 500 ml beaker filled with well-aerated pond water was pre-chilled and maintained at 4 °C. This beaker served as the cooling apparatus. Snails were placed in the cooling apparatus for 10 min at various times after conditioning. In control experiments it was found that snails resumed eating behavior within 2 minutes of being placed into the test chamber maintained at room temperature (i.e. 22 °C). Thus, we always waited for about 10 min following their removal from the cooling apparatus before testing their response to the CS.The first experiment was designed to determine whether 20 paired CS-US presentations given over 4 days resulted in better learning and memory formation than 20 paired CS-US presentations given over either 1 or 2 days.We then examined whether the cold-block procedure interfered with memory formation. In the 4-day training procedure, snails were subjected to a cold-block immediately after the 5th CS-US pairing each day.Experiment 2 was designed to elucidate the critical time following training during which the cold-block prevents STM and LTM formation. We applied the cold-block at 4, 7, 10, 15, or 180 min after training. We then examined whether memory had formed by testing the response to the CS immediately after the cold-block and at 24 h and 48 h later. When the cold-block was applied with a 4-min delay, animals were placed into the chilled water for 10 min starting at 4 min post-conditioning. Following the cold-block snails were kept in the test chamber at room temperature (~22 °C) for at least 2 min before the “after-cooling immediate post-test” was performed.Thus, each snail received the CS first as the pre-test, then the same number of CS-US pairings, the cold-block for 10 min at the various specified times following conditioning. In addition, they each were presented with the CS 3 times following conditioning at different times in order to determine if memory formed. The times were as follows: 1) immediately after the cold-block for STM; 2) 24 and; 3) 48 h after the conditioning for LTM. In the case of 15 min or 180 min experiments, we examined snails’ feeding response at 10 min after the final CS-US pairing as a post-test before subjecting the snails to the cold-block. To test the cold-block’s effect an immediate post-test was applied immediately after the cold-block. Thus, the post-test in 15 min and 180 min delay experiments was applied to snails twice (10 min and 27 min) after the final CS-US pairing. In experiment 2 we used 48 snails. The timing of each post-test session is shown in th to the 8th day these snails were again conditioned but without cold-block, i.e., they received 5 pairings of CS-US presentations for a further 4 days. They received a CS on the 5th day (i.e. following the 4 days of pairing and cold block) prior to next 4 days of CS-US pairing without the cold block. They all received an immediate post-test on each of the next 4 days of pairing as well as a 24 h and 48 h post-test.In this experiment, we examined whether cold-block-induced amnesia was reversible, in contrast to certain pharmacologic treatments. That is, we examined whether snails that were subjected to the cold-block procedure were still capable of forming LTM. For these experiments 7 snails were received 5 repeated pairings of the CS-US followed by an immediate cold-block, i.e., 0 min-delay, every day for 4 days. From the 5To test the hypothesis that STM formation is dependent on PKC activation, the PKC α and ε activator bryostatin was administered to the snails. Snails were placed in bryostatin-containing (0.5 ng/ml) pond water at 22°C for 45 min before the last day of training (5 CS-US pairings/day for 4 days). In these experiments, the cold-block was applied immediately after the final training session each day. STM and LTM were evaluated by an immediate post-test and a 24-h post-test, respectively.Hermissenda [Lymnaea [The PKC activator bryostatin  was initially dissolved in 100 µl ethanol and diluted in pond water to make a 1 µg/ml stock solution. This solution was further diluted in pond water to a final concentration of 0.5 ng/ml, with a final ethanol content of 0.005%. This was the maximum upregulating concentration of bryostatin found to be effective in the mollusk missenda ,44 and L[Lymnaea . AnimalsF post hoc test was then used to further determine the statistical significance of the differences between groups by KaleidaGraph version 4.0 . Both the behavioral measurements and the analyses were performed by researchers blind to the behavioral manipulation of the animals.Behavioral differences between pre- and post-conditioning  were tested with repeated-measures analysis of variance (ANOVA). Scheffe’s We previously demonstrated that 20 paired CS-US presentations was the minimum number of pairings required for LTM formation . Here wep<0.05). The 24-h post-test sessions in the other two groups were not significantly different, however STM was acquired in every groups . That is, the number of bites in the pre-test session in response to the CS was significantly greater than the number of bites elicited by the CS in the 24-h post-test sessions . The groups receiving the cold block 10 and 15 min after the final pairing showed both STM and LTM formation . Neither STM nor LTM was observed. Before beginning the day 4 training, we placed the snails in bryostatin-containing pond water at 22 °C for 45 min and then proceeded to train the snails (5 CS-US pairings). This training procedure should not have resulted in STM, however STM (significant feeding suppression after the 5 pairings within a day) was observed even though the cold-block was applied immediately after the 5Lymnaea; 57 snails for Experiment 1; 40 snails for Experiment 2; 7 snails for Experiment 3; 6 snails for Experiment 4. The findings of the present study confirmed that: 1) Spaced learning for TAC is more effective for inducing the formation of LTM; 2) A cold-block procedure prevents the formation of both STM and LTM if applied without delay of the last CS-US pairing on each day’s training; 3) A cold-block procedure prevents the formation of LTM if applied within 10 min of the last CS-US pairing on each day’s training; 4) A time delay of 180 min following the last CS-US pairing each day leads to no memory saving ; 5) The cold-block procedure, while sufficient to block both STM and LTM formation, did not permanently prevent snails from learning and forming both STM and LTM; and 6) PKC activity appeared to enhance memory formation.In this study we examined 110 c-fos, c-jun, and the recently identified target gene insulin-like growth factor 2 [Memory can be parsed into three categories based on its duration and molecular underpinnings. STM persists for minutes, ITM persists for a few hours, and LTM persists for days to months to years. STM does not require new protein synthesis whereas both ITM and LTM require new protein synthesis. In addition, LTM requires altered gene activity ,48–54. Afactor 2 ,55–60.Lymnaea is CCAAT/enhancer binding protein [Lymnaea [One of the known immediate early genes cloned and sequenced in TAC in  protein  and rece[Lymnaea .Lymnaea, Fulton et al (2005) reported that one-trial appetitive conditioning data suggest that there is a single period of sensitivity for the protein synthesis necessary for LTM that lasts from between 10 min to 1 h after training, based on studies using pharmacologic agents (the translation inhibitor anisomycin and the transcription blocker actinomycin D) [Lymnaea [Protein synthesis is essential for LTM formation. Two periods of sensitivity are assumed; one occurring immediately after training  and the mycin D) . Fulton on et al 005 repor[Lymnaea . As demoCooling snails immediately  after operant conditioning training, reactivation of memory, or extinction training blocks the consolidation or reconsolidation process and does not adversely affect the health of the snails ,63. In tThus, our findings using the cold-block procedure are consistent with the previous findings ,3. Here,Lymnaea, and in Hermissenda and Aplysia. In the Lymnaea study, which examined appetitive one-trial food conditioning, memory could be demonstrated at 10 min, 1 h, and 3 h, but not at 30 min or 2 h after a single training episode [Hermissenda study by Crow and Xue-Bian, changes in the protein levels at two different times following one-trial in vitro conditioning were examined using two-dimensional difference gel electrophoresis. Significant protein regulation was detected at 30 min and 3 h post-conditioning. These proteins were involved in diverse cellular functions, such as translational regulation, cell signaling, cytoskeletal regulation, metabolic activity, and protein degradation [Aplysia studies, memory at the behavioral level and its synaptic correlates were not observable at various times after training [Aplysia study, however, did not block STM as it did here. Finally, following a one-trial training procedure for operant conditioning of aerial respiration, ketamine blocked LTM formation if applied either immediately or 2 h after training [To further complicate matters, we found that applying the cold-block 180 min after the last CS-US pairing each day blocked memory formation entirely, which was an unexpected finding. It could be argued that STM was not observed because once formed it only persisted a short period of time and had dissipated prior to testing 180 min after the last CS-US pairing and after the cold-block procedure. Why LTM was not apparent on the last day following this procedure remains an important question because it appears that the consolidation period should have been completed within 10 min after each day’s training. A somewhat similar phenomenon was recently been reported in  episode . Moreoveradation . In the training ,70. Intetraining . In theitraining . The autFinally there is another possibility. We cannot rule out the possibility that the experimental procedure used in the “180 min delay cold-block” induced extinction of the learned response. With the “180 min delay cold-block” the CS alone was applied twice (10 min and 27 min) after the last CS-US in order to test for STM before the cold-block. The extra CS application may initiate an extinction process and this process might be enhanced during a cold-block period, thus no LTM was observed.At the cellular level, memory is evidenced by a change in synaptic activity at the pre-and/or post-synaptic neurons ,71,72. CHermissenda, a mollusk related to Lymnaea, classical conditioning  results in a change in the excitability of type B photoreceptors due to long-lasting K+ channel inactivation [Hermissenda, bryostatin enhances memory formation via a PKC pathway [Lymnaea as shown in Experiment 4. These data are consistent with data from operant conditioning studies in Lymnaea showing that bryostatin enhances long-lasting memory formation [Lymnaea should focus on examining changes in the excitability of neurons such as RPeD11 [In tivation ,76. In H pathway . Here weormation . Future s RPeD11 –80 to deHermissenda [The data presented here are also consistent with the finding reported by Epstein et al. in 2003 that the protein synthesis required for ITM and LTM occurs within 15 min following conditioning and again after 60 min, suggesting that a second round of protein synthesis is required for memory following classical conditioning in missenda . Here weLymnaea were successfully conditioned with 20 paired presentations of a sucrose stimulus as the CS and a tactile stimulus to the head as the US. The most effective training procedure leading to LTM formation was a ‘spaced’ procedure, comprising 5 paired CS-US presentations each day for 4 days. This memory persisted for more than 1 week. Application of a cold-block immediately was sufficient to disrupt both STM and LTM. Delaying the cold-block procedure by 10 to 15 min allowed for the formation of STM and LTM. Delaying the cold-block by 180 min following the completion of training also blocked both STM and LTM formation, suggesting that there are two critical periods for LTM formation. The initial period may correspond to macromolecular protein synthesis while the 180-min period may correspond to mRNA transcription or a second wave of protein synthesis.


Very few information is available about the changes of nitrogen metabolism occurring under Fe deficiency in Strategy I plants. The aim of this work was to study how cucumber (Cucumis sativus L.) plants modify their nitrogen metabolism when grown under iron deficiency.Nitrogen is a principal limiting nutrient in plant growth and development. Among factors that may limit NOThe activity of enzymes involved in the reductive assimilation of nitrate and the reactions that produce the substrates for the ammonium assimilation both at root and at leaf levels in Fe-deficient cucumber plants were investigated. Under Fe deficiency, only nitrate reductase (EC 1.7.1.1) activity decreased both at the root and leaf level, whilst for glutamine synthetase (EC 6.3.1.2) and glutamate synthase (EC 1.4.1.14) an increase was found. Accordingly, the transcript analysis for these enzymes showed the same behaviour except for root nitrate reductase which increased. Furthermore, it was found that amino acid concentration greatly decreased in Fe-deficient roots, whilst it increased in the corresponding leaves. Moreover, amino acids increased in the xylem sap of Fe-deficient plants.aa, organic acids) to the leaves.The data obtained in this work provided new insights on the responses of plants to Fe deficiency, suggesting that this nutritional disorder differentially affected N metabolism in root and in leaf. Indeed under Fe deficiency, roots respond more efficiently, sustaining the whole plant by furnishing metabolites (i.e.  It has  [IRT1])-6. The FArabidopsis[3- uptake has been documented. In fact, a decrease in NO3- uptake was observed in Fe-deficient cucumber plants[3- assimilation and Fe acquisition could compete for reducing equivalents; thus a limitation in NO3- uptake could favour the reduction-based mechanism of Fe uptake[3- reduction is carried out by Fe-containing enzymes (i.e. NR and NiR), Fe deficiency could alter the cytosolic NO3- concentration leading to a restriction of its uptake mechanism(s).However, there is fragmentary information about the change in N metabolism that occurs under Fe deficiency-11. Fromabidopsis-14. The r plants. Wang etr plants found the uptake or, alte3- assimilation requires reductants to be supplied to both NR and NiR. However, if NR activity is directly limited by the level of NADH in plants remains an open question[3- reduction and mitochondrial electron transport may compete each other for reductants under some conditions. In addition to NO3- reduction, a second process that may be subject to redox modulation in the C/N interaction is the formation of organic acids. Organic compounds such as 2-oxoglutarate (2-OG) act as carbon skeleton acceptors of amino groups in the biosynthesis of amino acids and their production requires oxidation through the respiratory pathways[At the metabolic level Fe deficiency induces several changes mainly concerning carbon (C) metabolism-20. C anquestion. Kaiser question,33 reporpathways,34-36. Opathways. As aminpathways and the CsFRO1, CsHA1 and CsIRT1 increased in response to the lack of Fe in agreement with data reported in the literature[In this work the change in the N metabolism in response to Fe deficiency has been studied at root and leaf levels. We have re-examined the responses to Fe starvation in cucumber plants previously grown for 7 d in the presence of Fe and, after this period, deprived of the micronutrient. Figureterature,40.3- both in roots and in leaves. No significant differences among treatments were found for roots, while a decrease (−35%) was found in the leaves in all the days assayed after induction of Fe deficiency. The NR activity, which is the first enzyme in the NO3- assimilatory pathway, decreased both at root and at leaf level during the progression of the Fe deficiency-induced condition . However, in Fe-deficient leaves, NR activity decrease was much more evident as compared to the root reaching −80% after 7 d, suggesting that Fe deficiency affected more the NR activity in the leaves than in the roots  at 7 d  concentration in roots and leaves of plants during the progression of Fe deficiency. The analysis of aa closely related to glycolysis and to the Krebs cycle, like, glutamate (Glu), glutamine (Gln), aspartate (Asp), asparagine (Asn), arginine (Arg), glycine (Gly), and serine (Ser) was chosen. In general, it is possible to note that aa concentration decreased in Fe-deficient roots, while it increased in leaves, as the Fe deficiency goes forward, with the sole exception of the Arg  while it increased in Fe-deficient leaves (+125%). The Asn concentration differed at leaf level where the increase was about 13-fold at 7 d of -Fe condition compared to the control, while it did not show any difference in the roots . At the same time also the citrate concentration was increased in the xylem sap at d 7 (+50%), confirming the data obtained by Abadía and co-workers[3- in the xylem was slightly decreased (−14%). The data collected from xylem sap are considered as an amount increase in nitrate, citrate and aa, since transpiration (E) rate did not change during the expression of Fe deficiency  and the aspartate-aminotransferase (AST). AST was also considered, along with ICDH, as one of the main candidates in the synthesis of 2-OG[We have assayed the activity of two enzymes that are recognised to be involved in the recycling of  of 2-OG. As showaa biosynthesis[aa biosynthesis downstream the GS/GOGAT cycle activity.It is well documented that Fe deficiency strongly affects plant C metabolism altering all metabolic pathways directly or indirectly related with it,38,42. Nynthesis. In agreynthesis,44. MoreIn this work we have determined both the activity and the gene expression of GS and GOGAT at root and leaf levels. Under Fe deficiency, GS activity increased in roots whilst it did not show any substantial difference in leaves Figure. The inc3- reduction (mediated by NR) decreased in both tissues under the same condition  a major utilization of aa in the protein synthesis, (ii) an increase in their translocation to the leaves and (iii) a degradation and/or recycling of aa. Direct evidence supporting the first hypothesis have been documented in Fe-deficient cucumber root[aa concentration in the xylem sap was observed during the progression of Fe deficiency  via xylem from root to leaf. Thus, under Fe deficiency, root seems to respond more efficiently sustaining the whole plant by furnishing metabolites  to the leaves. A schematic representation of the features of the metabolic pathways is reported in FigureIn conclusion, it is suggested that Fe deficiency strongly affected N metabolism by limiting NR activity and by increasing GS/GOGAT, in both root and leaf. Nevertheless under Fe deficiency root and leaf showed some important differences: the Cucumis sativus L. cv Marketmore ‘76, F.lli Ingegnoli, Milano) were surface sterilized and sown in Agriperlite, watered with 0.1 mM CaSO4, allowed to germinate in the dark at 26°C for 3 d, and then transferred to a complete nutrient solution with 0.1 mM Fe(III)-EDTA. Growing conditions were as reported by Rabotti and Zocchi[Cucumber seeds . Measurements were carried on fully expanded, intact leaves of Fe sufficient and Fe deficient plants. Net COTaq DNA polymerase (Promega) and the identity of the amplified fragments verified by sequencing. Fe-deficient and control tissues were pulverized in liquid nitrogen using a mortar and pestle and total RNA was extracted using Trizol Reagent (Invitrogen). Thirty micrograms of total RNA per lane were separated by electrophoresis at 5 V cm2 in a 1.3% (w/v) agarose gel containing 6% (v/v) formaldehyde, transferred to a Hybond-N1 nylon membrane (Amersham Bioscience) by capillary blotting in 20X SSC and then fixed by UV cross-linking. The blot was hybridized with a 32P-labeled cDNA probe for the entire coding sequence of all the genes considered in the study. Pre-hybridization and hybridization were conducted according to the nylon membrane manufacturer’s instructions. The membrane was washed for 10 min with 23 SSC in 0.1% (w/v) SDS at room temperature, with 13 SSC in 0.1% (w/v) SDS at 65°C for 20 min and then for 10 min with 0.13 SSC in 0.1% (w/v) SDS at 65°C.RNA was extracted from root and leaf tissues according to Vigani et al.. The genin silico analysis performed using the information available in the following databases:The probes for the genes considered in this work were designed after an http://www.brenda-enzymes.org;BRENDA,http://www.ncbi.nlm.nih.gov/;NCBI,http://www.arabidopsis.orgTAIR,We used PCR product fragments as cDNA probes obtained by amplification of the primers as reported in Additional filehttp://www.icugi.org/cgi-bin/ICuGI/tool/blast.cgi), the probe sequences obtained displayed percentage identity values against Cucurbit genes as reported in Additional fileCucumis melo (accession n. AY773090).After a Blast analysis in the Cucurbit genome database CuGenDB  analysis was performed on 0, 1, 3, 7-d plants. Leaves and roots were harvested separately. Free aa were extracted from fresh tissues at 4°C, first in 80% ethanol over night, then in 60% ethanol for 1 h and finally in distilled water for 24 h. The supernatants of each sample were pooled, aliquoted and kept at −20°C. Free aa were determined by HPLC as described by Muller and Touraine[Amino acid . Nitrate concentration was determined according to. Citraterding to.Total protein concentration was determined by the using dye-binding method of Bradford, using γt test at the P≤0.05 level in all cases.All statistical analyses were conducted with Sigma-Stat® 3.1. Means were compared by Student’s aa: Amino acid; ALT: Alanine aminotransferase; Arg: Arginine; Asn: Asparagine; Asp: Aspartate; AST: Aspartate amonitransferase; G-3-P-DH: Glyceraldehyde 3-phosphate dehydrogenase; Glc-6P-DH: Glucose 6phosphate dehydrogenase; Gln: Glutamine; Glu: Glutamate; Gly: Glycine; GS: Glutamine synthetase; GOGAT: Glutamate synthase; ICDH: Isocitrate dehydrogenase; PEPC: Phosphoenolpyruvate carboxylase; Ser: Serine.The authors declare that they have no competing interests.AB carried out gene expression analysis, enzyme assays, amino acid analysis. GV carried out enzyme assays, collection and analysis of xylem sap, western blotting analysis, determination of physiological parameters and wrote the draft of the manuscript. GZ participated in the strategic planning of the work, data analysis and writing the final version of manuscript. All the authors contributed to the discussion of the results and took part to the critical revision of the manuscript. All authors read and approved the final manuscript.Enzyme activity expressed as as nmol NADPH mg-1 prot min-1.Click here for fileConcentration of amino acids.Click here for fileWestern Blot analysis of nitrate reductase (NR) was performed on soluble fraction extracted from cucumber roots. 0, 1, 3, 7 are the days after Fe withdrawing.Click here for fileOligonucleotide primers for RT-PCR.Click here for file


Corrugation and scrolling exist dominantly in the modified GONs. GONs were single layered with the maximum solubility in n-octane up to 3.82 mg mL-1. Oleylamine molecules chemically attach onto the GONs. Compared with traditional strategies, the phase transfer method has the features of simplicity and high efficiency.Phase transfer method has been developed for preparing homogeneous and stable graphene oxide colloids. Graphene oxide nanosheets (GONs) were successfully transferred from water to  Graphene has attracted considerable attention over the past few years due to its exceptional properties -3, such -1 [-1, which was more than twice of that for bare graphene oxide nanosheets. When p-phenyl-SO3H groups were introduced into the graphene oxide, the resulting reduced product remained soluble in water and did not aggregate [N-methyl-2-pyrrolidone (NMP), but not in N,N-dimethylformamide (DMF) [l-lysine as a linker through a covalent amide group [Several approaches have been proposed for the production of aqueous suspensions of graphene oxide and graphene sheets. Exfoliation of graphite oxide either by rapid thermal expansion or by ultrasonic dispersion has been widely adopted to prepare graphene oxide and graphene sheets in bulk. Due to the rich hydrophilic oxygen-containing groups such as carboxyl, hydroxyl, and epoxide, the graphite oxide readily suspends in water and polar organic solvents, such as ethylene glycol, DMF, NMP, and THF at about 0.5 mg mL-1 . In orde-1 . The maxggregate . Phenylede (DMF) . Triblocde (DMF) . Water-sde group . KOH coude group , and exfde group ; therefoMost of the reported studies are related to the aqueous graphene oxide and graphene suspensions. Organophilic graphene oxide and graphene nanosheets are important for the application in graphene-based composite materials, while only a few papers presented the methods to produce hydrophobic graphene oxide and graphene nanosheets . For exaFor preparing the suspensions containing graphene or graphene oxide nanosheets (GONs), the traditional strategy is a three-step method. First, graphene or graphene oxide nanosheets are prepared and dried, and then they will be modified. Finally, the functionalized graphene will be dispersed in solvents under stir or ultrasonication. The three-step method is used widely, while there are some drawbacks. First of all, during the synthetic and drying process, GONs have the strong trend to conglomerate due to the large surface area. Second, not all the GONs can be modified, and there is always some sediment under the bottom of suspension. The suspension is not homogeneous, which is not desired for preparing even films and composite materials. Here, we developed a facile phase-transfer method with high efficiency to prepare stable suspensions of graphene oxide in organic solvents. This method is primarily based on the strong interaction between GONs and oleylamine.2SO4, 30% H2O2, potassium permanganate (KMnO4) and n-octane were obtained from Shanghai Chemical Reagents Company. Oleylamine was purchased from Aldrich. All other reagents were used without further purification.Natural graphite was purchased from Qingdao Baichuan Graphite Co., LTd. 98% Hn-octane was added into the mixture. The phase transfer process occurred spontaneously, and there was a distinct phase interface between the aqueous and octane in 1 day  was used for thermogravimetric analysis and calculation of the decomposition activation energy . The UV–Vis spectra of the octane suspensions containing GONs were measured on a Shimadzu UV2550 UV–Vis spectrometer. In order to determine the concentration of the suspension, the calibration line was constructed by measuring UV–Vis spectra absorption intensities of the suspensions at different concentrations (0.1–0.5 mg mL-1). The standard solution with 0.5 mg mL-1 was prepared through the following process. At first, 20 mL of 0.5 mg mL-1 aqueous solution containing GONs was prepared, then 50 mg oleylamine was added, after that GONs-OA would be transferred to 20 mL octane, and the resulting was the octane suspension containing GONs with the concentration 0.5 mg mL-1. To determine the maximum solubility of the suspension, the supernatant solution of the saturated suspension was diluted several times for the measurement of UV–Vis spectra until the absorbance fitting in the range of calibration line. Based on the calibration line, the concentration of the diluted solution could be determined. Finally, the maximum solubility could be obtained based on the times of the dilution.The size and morphology of the GONs were examined by using transmission electron microscopy . The TEM samples were prepared by dropping the diluted colloid onto a carbon film mesh supported on a copper grid and drying it in air. Atomic force microscopic (AFM) images were taken on a MultiTask AutoProbe CP/MT Scanning Probe Microscope . Imaging was done in tapping mode using a V-shaped 'Ultralever' probe B and nominal tip radius 10 nm. The diluted GONs colloid was dropped onto a freshly cleaved mica. After the mica was dry, the GONs-OA were imaged. FT-IR spectra were recorded with a Bruker Equinox V70 FT-IR spectrometer in dry KBr pullet in the range of 400–4,000 cmn-octane. The phase transfer process of preparing organosol was simple. The GONs modified by oleylamine could be transferred from aqueous solution to organic solvent easily with high efficiency. Almost all the GONs could be transferred to octane , C=O stretching of carboxyl groups situated at edges of GONs sheets , epoxide groups and skeletal ring vibrations  and the stretching vibration of C–O–C  [-1 in GONs-OA correspond to the C–H stretching vibrations in oleylamine. The amine group (N–H) is also observed with a band 1,458 cm-1 for GONs-OA, which corresponds to 1,465 cm-1 in the FT-IR spectrum of oleylamine  and νs(COO-), respectively. The facts illustrate that the –COOH groups of GONs is ionized to –COO-, and it clearly confirms that oleylamine is attached to GONs through chemical absorption.The phase transfer method is based on the strong interaction between GONs and oleylamine, which is confirmed by the FT-IR spectral analysis. Figure 84 cm-1) . Figure e Figure . Compare-NH3+ ionic bond. Li et al. investigated the extraction of maleic acid using trioctylamine, and they found the amine formed 1:1 complexation with maleic acid through ion pairing [Kahn prepared soluble oxidized single-walled carbon nanotubes (SWNT) in organic and aqueous solvents through derivatization using 2-aminomethyl-18-crown-6 ether (CE) . They pr pairing . Zhou et pairing , and the-1. The derivative thermogravimetric plots of GONs-OA at different heating rate are shown in Figure y = -29.64817 × 103x + 44.91489 (R2 = 0.997) and y = -28.24605 × 103x + 29.80937 (R2 = 0.997), respectively (Figure -1, respectively. The decomposition activation energy is large, also demonstrating the strong interaction between oleylamine and GONs.In order to further investigate the interaction of GONs and oleylamine, the decomposition activation energy of GONs-OA was investigated through the thermo-gravimetric analysis under nitrogen atmosphere with the heating rate 5, 10, 20, 30, and 40 K min2, and steam. For the TG curve of pure oleylamine, there is an obvious weight loss process with an onset temperature 145 °C, corresponding to the boiling point of oleylamine. GONs-OA displays different thermal behaviors. For GONs-OA, below 150 °C there is no weight loss. Because of the hydrophobic property of GONs-OA, polar molecules will not be absorbed on the surface. The onset temperature of GONs-OA is 178 °C, and the main mass loss is in the range of 280–520 °C with the peak at 425 °C. The process is due to the decomposition of the organic functional groups and further carbonization of the graphene backbone. The weight loss for GONs-OA from 50 to 800 °C is 71.7%, mainly due to the absorbed oleylamine molecules. The left 28.3% is graphene oxide. Through a crude calculation, we can obtain the molar ratio of graphene carbon and oleylamine, it is 8.8:1. Because GONs-OA were washed by hot ethanol for 5 times, oleylamine molecules would be absorbed on the surface by monolayer absorption. Therefore, it is estimated that about 11.4% carbon atoms of GONs is absorbed by oleylamine molecules.Figure n-octane at different weight concentration. Due to the absorbed oleylamine molecules, GONs-OA suspensions present the characteristic absorption band at 267 nm, assigned to the C=C of oleylamine. The band of GONs-OA at 267 nm is so strong as to shadow the typical absorption peak at 230 nm of GONs [n-octane at different concentration at the wavelength of 500 nm. The inserted figure in Figure -1. Wang et al. used the same method to obtain the solubility of hydrophobic graphene oxide nanosheets prepared via functionalizing with phenylisocynate through a solvothermal synthesis process, and the maximum solubility of GONs in DMF solution was determined to be 0.56 mg mL-1 [Figure  of GONs . Accordi mg mL-1 . Compari-1, respectively, indicating the strong interaction between oleylamine and GONs. The solubility of GONs-OA in octane was quantitatively characterized by UV–Vis spectroscopy, and the maximum solubility of GONs-OA in octane solvent was up to 3.82 mg mL-1.Optimizing the dispersion of graphene oxide in non-polar solvent is vital for the application and production of graphene-based composites. Synthetic methods for the colloidal suspensions containing graphene and graphene oxide are one of research hotspots. In this paper, the phase transfer method was applied to prepare homogeneous and stable suspensions containing GONs in non-polar solvents. Compared with the traditional three-step method, phase transfer strategy is very simple. Due to the strong interaction between oleylamine and GONs, almost all the GONs could be transferred to the organic phase with high efficiency. The size of GONs-OA was in the range of 300–800 nm, and GONs-OA always existed in the state of corrugation and scrolling. Almost all the sheets were single-layer graphene oxide, and the thickness of GONs-OA was uniform. Due to the presence of oleylamine absorbed on both sides of the GONs, the average thickness of GONs-OA was about 1.3 nm. The FT-IR spectra clearly confirmed that oleylamine molecules were attached to GONs through chemical absorption. The thermo-gravimetric analysis of GONs-OA illustrated that about 11.4% carbon atoms were absorbed by oleylamine. The decomposition activation energy calculated through Ozawa and Kissinger method were 234.31 and 234.84 kJ mol


Background. Psychodynamic psychotherapy is a psychological treatment approach that has a growing empirical base. Research has indicated an association between therapist-facilitated affective experience and outcome in psychodynamic therapy. Affect-phobia therapy (APT), as outlined by McCullough et al., is a psychodynamic treatment that emphasizes a strong focus on expression and experience of affect. This model has neither been evaluated for depression nor anxiety disorders in a randomized controlled trial. While Internet-delivered psychodynamic treatments for depression and generalized anxiety disorder exist, they have not been based on APT. The aim of this randomized controlled trial was to investigate the efficacy of an Internet-based, psychodynamic, guided self-help treatment based on APT for depression and anxiety disorders.Methods. One hundred participants with diagnoses of mood and anxiety disorders participated in a randomized (1:1 ratio) controlled trial of an active group versus a control condition. The treatment group received a 10-week, psychodynamic, guided self-help treatment based on APT that was delivered through the Internet. The treatment consisted of eight text-based treatment modules and included therapist contact  in a secure online environment. Participants in the control group also received online therapist support and clinical monitoring of symptoms, but received no treatment modules. Outcome measures were the 9-item Patient Health Questionnaire Depression Scale (PHQ-9) and the 7-item Generalized Anxiety Disorder Scale (GAD-7). Process measures were also included. All measures were administered weekly during the treatment period and at a 7-month follow-up.Results. Mixed models analyses using the full intention-to-treat sample revealed significant interaction effects of group and time on all outcome measures, when comparing treatment to the control group. A large between-group effect size of Cohen’s d = 0.77 (95% CI: 0.37–1.18) was found on the PHQ-9 and a moderately large between-group effect size d = 0.48 (95% CI: 0.08–0.87) was found on the GAD-7. The number of patients who recovered  were at post-treatment 52% in the treatment group and 24% in the control group. This difference was significant, χ2 = 8.3, p < .01. From post-treatment to follow-up, treatment gains were maintained on the PHQ-9, and significant improvements were seen on the GAD-7.Conclusion. This study provides initial support for the efficacy of Internet-delivered psychodynamic therapy based on the affect-phobia model in the treatment of depression and anxiety disorders. The results support the conclusion that psychodynamic treatment approaches may be transferred to the guided self-help format and delivered via the Internet. The aim of this randomized controlled trial was to investigate the efficacy of an Internet-delivered psychodynamic guided self-help treatment for depression and anxiety disorders that was based on the affect-phobia model of psychopathology . The proDepression and anxiety disorders are major world-wide health problems which lower the quality of life for the individual and generate large costs for society . LifetimPsychodynamic psychotherapy is a psychological treatment approach that has a growing empirical base , with reexperiential dynamic therapies (One psychodynamic treatment that has a strong focus on expression and experience of affect is affect-phobia therapy (APT), developed by herapies , which iherapies , and Accherapies . Two ranherapies . Howeverherapies , to dateDuring the last decade, numerous trials on guided self-help and Internet-delivered cognitive behavior therapy (CBT) for various psychiatric disorders have been conducted . For milThis randomized controlled trial aimed to examine the effects of an Internet-delivered psychodynamic treatment based on the affect-phobia model of psychopathology. Participants had depression and anxiety disorders. The treatment was given as self-help with additional therapist support via the Internet, and compared to a control group who also received online support. As compared with the control condition, a significant effect of treatment was expected both on measures of depression and anxiety for the full sample. In addition, a larger effect was expected on measures of depression for participants with depression as their main presenting problem as compared with those who did not have this as the main problem. Similar, a larger effect on anxiety measures was expected for participants with a principal anxiety diagnosis as compared with those who did not have such a diagnosis. We also investigated the uncontrolled effects of the treatment 7 months following the completion of the treatment.This study is reported in accordance with the CONSORT statement for clinical trials . The cliPatients were recruited via the Internet and advertisements in newspapers during January 2012. The final follow-up evaluation occurred in December 2012. Patients were eligible for participation if they (1) had at least one of the following Axis-I diagnoses, specified by DSM-IV criteria: Major depressive disorder, social anxiety disorder, panic disorder, generalized anxiety disorder, depressive and/or anxiety disorder not otherwise specified; (2) had a raw score of at least 10 on either the 9-item Patient Health Questionnaire Depression Scale PHQ-9;  or the 7After initial application, participants were invited to an online screening which consisted of demographic questions and online versions of the outcome measures (see below). These results were later used as a pre-treatment assessment. If initial inclusion criteria were met (having more than 10 on the PHQ-9 or the GAD-7), participants were contacted for a telephone-based diagnostic interview, based on the Mini-International Neuropsychiatric Interview MINI; . This prThe treatment lasted for 10 weeks and consisted of eight self-help modules given with text-based therapist support. A secure online environment was used both for the delivery of self-help material and for communication with the therapists. Therapist support was given asynchronously, i.e., similarly to e-mail. The primary role of the therapists was to give feedback on completed modules and administer gradual access to the treatment. In general, feedback was given on Mondays, but the therapists were available to answer additional questions within 24 h.The self-help modules were based on the book ‘Living Like You Mean It’ by Ronald J. Frederick  that folFor ethical reasons, participants on the waiting list also had continuous contact with an assigned therapist during the same 10-week period. Every Monday, therapists were scheduled to initiate contact with the participants, using the same secure online environment as used with the treatment group. Contact involved clinical monitoring of symptoms and questions typically regarding clients’ experiences from the previous week. Therapists were instructed to give basic support, but not to use any specific psychological techniques other than empathic listening and asking further questions. As the control group did not work with any treatment modules, the therapists were expected to spend less time with the participants from this group. After the treatment period had ended, participants from the control group were offered an 8-week version of the treatment. The results from that treatment period are, however, outside the scope of this study.r = .84  and the anxiety dimension of SCL-90 (r = .74)  . Both meTwo measures were included to assess two processes assumed to be relevant during treatment. Both measures were administered pre-treatment, weekly during treatment, post-treatment and at follow-up. The Emotional Processing Scale EPS-25;  was usedDSM-IV diagnoses, including a participant’s principal diagnosis, were recorded using the MINI Interview . This inThe therapists were three Master’s level students in their last semester of a 5-year clinical psychologist program. All therapists have had clinical training in affect-focused psychodynamic psychotherapy and had clinical experience from working with this kind of psychotherapy. Prior to the study, all therapists were also trained in providing guided self-help treatments via the Internet. Throughout the trial, clinical supervision was provided by psychologist Ronald J. Frederick, who had authored the original treatment manual. Treatment integrity and adherence to the treatment manual were monitored during supervision.To investigate differential efficacy between participants who had either depression or anxiety as their main presenting problem, all participants were classified based on their main symptomatology. The classification was based on the assessment of a participant’s principal diagnosis that was recorded in the diagnostic interview conducted at baseline. These categories were used to assess whether the treatment was more effective in treating depressive symptoms among participants with principal depression, and analogously regarding anxiety.2-tests  and independent t-tests (for continuous variables). Normality was confirmed by the Shapiro-Wilk test . That analysis suggested that 51.3 participants per group were needed to obtain the desired effect.Sample size was determined a priori based on power analyses. These power calculations were based on a linear mixed-effects model (10 time points with an autoregressive error structure with a random intercept and slope), an alpha set at 0.05, power set at 0.80, a predicted effect size of Cohen’s d) were calculated by dividing the differences in means by the pooled standard deviations . Additionally, there were no significant differences between the groups on any demographic data or current/past treatment with medication and/or psychological treatment. A complete description of demographic data of included participants is available in One hundred individuals with depression and/or anxiety disorders were enrolled in the study. There were no significant pre-treatment mean differences between the treatment group and the control group on any outcome measures  = 39.4, p < .001, with 55/57  compared to 17/43 (39.5%) for subgroups of depression and anxiety, respectively. Similarly, there were significantly more participants with a principal anxiety disorder that had GAD (67.4%) compared to 35.1% from the depression subgroup, χ2 = 10.3, p < .001. There were no differences between subgroups regarding diagnoses of panic disorder and social phobia. Also, there were no differences in any demographics. However, there was a significant difference between subgroups in depression severity as measured by the PHQ-9 at baseline, t(98) = 3.70, p < .001. However, no significant baseline difference on the GAD-7 was found t(98) = 1.23, p = .22.Regarding subgroups of principal depression and anxiety, there was a difference between subgroups in the number of participants in an acute episode of depression, χAt post-treatment, 100% of the data was collected. At the 7-month follow-up, 47/50 (94%) of the self-report measures and 40/50 (80%) of the data from the follow-up interviews  were collected. Adherence to treatment was defined as the number of modules completed. A module was only considered completed if the homework assignment had been sent to the therapist. Out of the 50 participants receiving treatment, 42 (84%) completed all modules. Only 4 participants (8%) completed less than half of the program.F = 19.94, p < .001, and the GAD-7, F = 7.86, p < .01. Both interaction effects were significant at the Bonferroni-corrected alpha level of p < .0125. Estimates of fixed effects for the PHQ-9 and the GAD-7 were as follows. Intercept: 12.37 and 10.71 (p’s < .001); Group: 0.11 and 0.14 (p’s > .85); Time: −0.64 and −0.49 (p’s < .001); Group × Time: 0.40 and 0.23 (p’s < .01). Random effects for the PHQ-9 and the GAD-7 were estimated as follows. Intercept: 14.33 and 12.78 (p’s < .001); Time: 0.15 and 0.12 (p’s < .001). Between-group effect sizes at post-treatment was large (d = 0.77) for depression and moderate (d = 0.48) for anxiety, favoring treatment over control. The continuous within-group changes on the PHQ-9 and the GAD-7 are illustrated in t-tests conducted post hoc showed that there were significant post-treatment versus follow-up decrease on the GAD-7, t(46) = 2.03, p < .05, and a trend towards a significant decrease on the PHQ-9, t(46) = 1.42, p = .16. For the EPS-25 and the FFMQ, there were also significant interaction effects of treatment group and time  = 26.5 and F = 29.9, respectively; Both p’s < .001). Both these interaction effects were significant at the Bonferroni-corrected alpha level of p < .0125. Fixed effects for the EPS-25 and the FFMQ were estimated as follows. Intercept: 4.70 and 72.76 (p’s < .001); Group: −0.16 and 3.67 (p’s > .15); Time: −0.18 and 1.39 (p’s < .001); Group × Time: 0.13 and −1.17 (p’s < .001). Estimates of random effects for the EPS-25 and the FFMQ were as follows. Intercept: 1.25 and 156.37 (p’s < .001); Time: 0.013 and 0.98 (p’s < .001). The between-group effect at post-treatment was large for the EPS-25 (d = 0.82) and moderate to large (d = 0.65) for the FFMQ.Means, standard deviations and effect sizes within and between groups for the self-report measures are presented in 2 = 7.3, p < .01). Reductions in the number of diagnoses of GAD, SP or PD were not significantly different between groups at post-treatment.The number of diagnoses among participants at pre-treatment, post-treatment and at the 7-month follow-up are illustrated in n = 26; 52.0%) and the control group , χ2 = 8.3, p < .01. At follow-up there were 25 participants (50.0%) from the treatment group who met the criteria for recovery.Categorical rates of recovery after treatment  were significantly different at post-treatment between the treatment group  = 12.1, p < .001. At follow-up, this figure was 52% (n = 26) in the treatment group.Post-treatment interviews resulted in estimates of clinical global improvement according to the CGI-I . In the d = 3.10) on the PHQ-9 in the depression subgroup, compared to for those in the anxiety subgroup (d = 1.12). Thus, there were no indications that the treatment was more effective in reducing symptoms of depression among participants with a principal diagnosis of depression, or analogously for anxiety symptoms.There were no significant interaction effect of group, subgroup and time on neither the PHQ-9 nor the GAD-7. This was despite the fact that the treatment had a very large within-group group effect (SD = 4.0). While there was a significant difference in average therapist time per week between therapists  = 7.73, p < .001), there were no correlations between therapist time and change scores on any of the outcome measures . The average therapist time per client and week was 2.3 min (SD = 0.86) in the control group. As expected, therapist time was significantly less in the control group than in the treatment group, t(98) = 12.4, p < .001.In the treatment group, the average therapist time per client and week was 9.5 min (Most participants were satisfied (46%) or very satisfied (36%) with the overall treatment they had received. Nine (18%) were indifferent or mildly dissatisfied, and no one was clearly dissatisfied. An absolute majority (82%) thought that the amount of text was appropriate. A similar amount of participants considered the text interesting and relevant, all the time (46%) or most of the time (40%). Most participants considered the treatment to be very demanding (28%), demanding (42%) or somewhat demanding (26%). Importantly though, a majority considered the treatment very much worth the effort (52%) or worth the effort (38%).This randomized controlled trial aimed to evaluate the effects of affect-phobia therapy in the format of guided self-help through the Internet in a sample of participants with depression and anxiety disorders. The results indicated that the treatment was effective in reducing symptoms of depression and anxiety, and also in facilitating emotional processing and mindfulness skills. Subgroup analyses gave no indications of differential efficacy between participants with a principal diagnosis of depression and those with principal anxiety. Treatment gains were maintained in the 7-month follow-up.The treatment manual used in this study aimed to implement a psychodynamic treatment based on the affect-phobia model in self-help format. This approach calls for a discussion on similarities and differences to the original APT manual. An assumption of this implementation was that the core principles of affect-phobia treatment manual could be retained. This included the general model of psychopathology (i.e., as illustrated by the triangle of conflict in d around 1.0 for measures of depression and anxiety. Hence, the affect-phobia treatment tested in this study, seems to stand well when compared to other transdiagnostic treatments tested.In affect-phobia therapy, the model of psychopathology is the same across disorders, i.e., the triangle of conflict is assumed to explain both etiology and maintenance of for example depression and anxiety disorders . This aiThere are methodological limitations that need to be considered. First, as we recruited participants from the community and not from, for example, a treatment clinic, the external validity of the findings are challenging to interpret. While there are studies on ICBT that suggests generalizability to clinical settings e.g., , this haThis study provides preliminary support for the efficacy of Internet-delivered psychodynamic treatment based on the affect-phobia model in the treatment of depression and anxiety disorders. This study provides further evidence that psychodynamic treatment approaches may be transferred to the guided self-help format and delivered via the Internet. Hence, this study adds to the empirical base of Internet-delivered psychological treatments and to that of psychodynamic psychotherapy in general. Finally, as we have no reason to believe that the treatment would perform less effectively in a face-to-face setting, the findings from this study call for further research on affect-focused psychotherapies.10.7717/peerj.102/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.102/supp-2Supplemental Information 2Click here for additional data file.